Compositions and methods for enhanced bacterial exopolysaccharide production

ABSTRACT

The present invention provides nucleic acid sequences and variants thereof capable of modulating exopolysaccharide production in  Sphingomonas , and provides methods of using such nucleic acid sequences to generate bacteria that hyper-produce exopolysaccharide in slime form.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the production ofexopolysaccharides, and more specifically, to a nucleic acid sequenceand variants thereof capable of modulating exopolysaccharide production,and to the use of such nucleic acid sequences to generate bacteria thathyper-produce exopolysaccharide in slime form.

2. Description of the Related Art

There is an increasing demand for inexpensive and environmentallyacceptable gelling agents for industrial applications and for the foodindustry. Some exemplary industrial applications of gelling agentsinclude oil field drilling, adhesives, paints, animal feed, householdproducts, personal care products (e.g., shampoo, lotion), oral careproducts (e.g., toothpaste), pharmaceuticals, and the like. Someexemplary uses of gelling agents in the food industry include use inpudding, dairy products, pie filling, dressings, confectionery, sauces,syrups, and the like. The biotechnology industry has responded to thisdemand for gelling agents by increasing the availability of a variety ofbacterial exopolysaccharide products that are acceptable for commercialuse.

Bacterial exopolysaccharides are useful compounds as gelling orviscosity increasing agents because of their distinctive rheologicalproperties (e.g., resistance to shear, compatibility with various ioniccompounds, stability to extreme temperatures, pH and saltconcentrations). A variety of bacteria produce exopolysaccharidesparticularly useful as thickening or gelling agents. For example, agenus of bacteria that produces many types of exopolysaccharides isSphingomonas. A few such polysaccharides include gellan, welan, rhamsan,S-7, and S-88 (see, e.g., Pollock, J. Gen. Microbiol. 139:1939, 1993).The exopolysaccharides produced by Sphingomonas are referred to as“sphingans,” and at least three sphingans (gellan, welan, and rhamsan)are commercially produced by large-scale, submerged fermentation.

Many bacterial exopolysaccharide products offer a range of attractiveimprovements over synthetically produced materials, but they remainrelatively expensive to produce because of the costs associated withrecovery and purification of a desired product. Furthermore, conditionsthat allow for higher fermentation yields of exopolysaccharides alsoresult in increased broth viscosity, which thickening ultimatelyrequires higher energy input to effectively disperse oxygen andnutrients to allow sufficient bacterial growth in the fermentationbroth. That is, fermentations that provide higher exopolysaccharideyields have also resulted in correspondingly higher production costs.

Hence, a need exists for a better understanding of bacterialbiosynthesis of exopolysaccharide to aid in the identification ofbacteria that produce more exopolysaccharide, and that produceexopolysaccharide in a form that does not increase the viscosity of afermentation broth. In addition, a need exists for methods of making oridentifying such bacteria, which in turn would allow optimization ofexopolysaccharide production and yield under typical, industrialfermentation conditions. The present invention meets such needs, andfurther provides other related advantages.

BRIEF SUMMARY OF THE INVENTION

The present invention relates generally to the use of a nucleic acidsequence and variants thereof capable of modulating exopolysaccharideproduction, and to methods of using such nucleic acid sequences togenerate bacteria that hyper-produce exopolysaccharide in slime form.

In one aspect, the present invention provides an isolated nucleic acidmolecule comprising a sequence that remains hybridized under highlystringent conditions to a probe, wherein the probe consists of SEQ IDNO:1 or a complement of SEQ ID NO:1. In one embodiment, theaforementioned isolated nucleic acid molecules wherein said nucleic acidmolecules encode at least one polypeptide capable of alteringexopolysaccharide production in a Sphingomonas species. In relatedembodiments, the at least one encoded polypeptide comprises an aminoacid sequence that is at least 80% identical to SEQ ID NO:2, or theencoded polypeptide comprises an amino acid sequence of SEQ ID NO:2 withconservative amino acid substitutions, or the polypeptide comprises theamino acid sequence of SEQ ID NO:2, or the polypeptide consists of theamino acid sequence of SEQ ID NO:2. In still another embodiment, theinvention provides an isolated nucleic acid molecule comprising anucleotide sequence as set forth in SEQ ID NO:1 or a complement of SEQID NO:1. In yet other embodiments, any of the aforementioned nucleicacid molecules are DNA or RNA.

In another embodiment, the invention provides a recombinant expressionvector comprising at least one promoter operably linked to anyone of theaforementioned nucleic acid molecules. In still another embodiment, therecombinant expression vector expresses the modulator polypeptide as afusion protein comprising a polypeptide product encoded by a secondnucleic acid sequence, such as a tag or an enzyme. In certainembodiments, the recombinant expression vector has a regulated promoter.In still other embodiments, the recombinant expression vector is aplasmid. In yet another embodiment, the recombinant expression vector isplasmid X026 or plasmid X029 (ATCC PTA-5127). In one embodiment, therecombinant expression vector comprises at least one promoter operablylinked to a nucleic acid molecule that comprises a nucleotide sequenceas set forth in SEQ ID NO:1.

In still another embodiment, the present invention relates to a hostcell comprising any of the aforementioned recombinant expressionvectors. In certain embodiments, the host cell is a prokaryotic cell,such as a sphingan-producing bacterium or a Sphingomonas cell or asphingan-producing Sphingomonas bacterium. In other embodiments, thehost cell is a Sphingomonas bacterium capable of producing a sphingansuch as gellan, welan, rhamsan, diutan, alcalan, S7, S88, S198, andNW11. In still other embodiments, the host cell produces a sphingan incapsule form or slime form. In certain embodiments, the host cell isSphingomonas strain α252 (ATCC PTA-5128, Welarn Slime), X287 (ATCCPTA-3487, Gallen Slime), X530 (ATCC PTA-3486), Z473 (ATCC PTA-3485),X031 (ATCC PTA-3488), or; 127. In a related embodiment, the host cell isa xanthan-producing bacterium, such as a Xanthomonas bacterium and morespecifically Xanthomonas strains X59 (ATCC 55298), X55 (ATCC 13951),α287, α300, or α301. In certain embodiments, the polypeptide or fusionprotein expressed from the nucleic acid on the recombinant expressionvector alters the level of exopolysaccharide production in the hostcell.

In yet another aspect, the invention provides an isolated bacterium thatproduces exopolysaccharide, comprising a bacterium capable of producingexopolysaccharide in slime form even when expressing a polypeptideencoded by any of the aforementioned nucleic acid molecules and whereinthe bacteria are any of the aforementioned bacteria, including a mutantof Sphingomonas strain α027; or a mutant of Xanthomonas strains α287,α300, or α301. In other embodiments, the bacterium that producesexopolysaccharide in slime form when expressing at least one polypeptideencoded by any of the aforementioned nucleic acid molecules is strainα062 (ATCC PTA-4426), α063, α065, or α069 or Xanthomonas strains α449(ATCC PTA-5064), α485, or α525. In yet other embodiments, the inventionprovides an isolated bacterium selected from Sphingomonas strain α062(ATCC PTA-4426), α063, α065, or α069 and mutants or derivatives thereof,wherein the bacteria are capable of producing an exopolysaccharide inslime form even when expressing at least one polypeptide encoded by anyof the aforementioned nucleic acid molecules, and mixtures thereof ofsuch bacteria. In still other embodiments, the invention provides anisolated bacterium selected from Xanthomonas strain α449 (ATCCPTA-5064), α485, or α525, and mutants or derivatives thereof, whereinthe bacteria are capable of producing an exopolysaccharide in slime formeven when expressing at least one polypeptide encoded by any of theaforementioned nucleic acid molecules, and mixtures thereof.

It is another aspect of the invention to provide a method forhyper-producing exopolysaccharide, comprising culturing bacteria underconditions and for a time sufficient to permit exopolysaccharideproduction, wherein the bacteria hyper-produce exopolysaccharide inslime form when expressing at least one polypeptide encoded by any ofthe aforementioned nucleic acid molecules; and separating theexopolysaccharide in slime form from such a culture. In certainembodiments, the bacteria are Sphingomonas bacteria, such as thosecapable of hyper-producing an exopolysaccharide selected from gellan,welan, rhamsan, diutan, alcalan, S7, S88, S198, and NW11, includingSphingomonas strains α062 (ATCC PTA-4426), α063, α065 and α069. Incertain other embodiments, the bacteria are Xanthomonas bacteria, suchas those capable of hyper-producing xanthan, including Xanthomonasstrains α449 (ATCC PTA-5064), α485, and α525. Also provided are any ofthe aforementioned methods wherein the culturing comprises fermentation,or wherein the bacteria produce from about 20 grams to about 60 grams ofexopolysaccharide per liter of culture. In some embodiments, thefermentation is conducted from about 48 hours to about 96 hours at atemperature ranging from about 25° C. to about 35° C. In still otherembodiments, the invention provides any of the aforementioned methodswherein the separating of exopolysaccharide from the bacteria is byalcohol precipitation, such as by adding about 1 to about 1.5 culturevolumes of alcohol to the culture. In other embodiments, thefermentation culture will have a viscosity ranging from about 15,000 cpto about 40,000cp.

Turning to another aspect, the invention provides a method for makingbacteria capable of hyper-producing an exopolysaccharide in slime form,comprising contacting bacteria suppressed for production of anexopolysaccharide in slime form with a mutagen, wherein the bacteria (i)contain a recombinant expression vector comprising at least one promoteroperably linked to a nucleic acid molecule that encodes a polypeptideencoded by a nucleic acid molecule according to any one of claims 2 to4, and (ii) express a polypeptide of part (i) such thatexopolysaccharide production is suppressed; and identifying there frombacteria capable of hyper-producing exopolysaccharide in slime form inthe presence of a polypeptide capable of suppressing exopolysaccharideproduction. In certain embodiments, there is provided a method formaking bacteria that are capable of hyper-producing an exopolysaccharidein slime form, comprising (A) contacting (a) a mutagen with (b) bacteriathat are capable of producing an exopolysaccharide in slime form,wherein the bacteria (i) contain a recombinant expression vectorcomprising at least one promoter operably linked to a nucleic acidmolecule that encodes at least one polypeptide, wherein the nucleic acidmolecule is any of the aforementioned nucleotide sequences, and (ii)express said at least one polypeptide of (i) such that exopolysaccharideproduction is suppressed, under conditions and for a time sufficient toproduce mutagenized bacteria; and (B) identifying among said mutagenizedbacteria one or a plurality of bacteria that are capable ofhyper-producing exopolysaccharide in slime form. In one embodiment, themutagen used in this method is ethylmethane sulfonate and the bacteriaare Sphingomonas bacteria, such as those capable of producing anexopolysaccharide selected from gellan, welan, rhamsan, diutan, alcalan,S7, S88, S198, and NW11, including Sphingomonas strain α027.

In another aspect, the invention provides a bacterium produced by anyone of the aforementioned methods, including a mutant of Sphingomonasstrain α027, such as α062 (ATCC PTA-4426), α063, α065, or α069, and amutant of xanthomonas strain α300, such as α449 (ATCC PTA-5046), α485,or α525.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relation between living cell number and OD₆₀₀ ofSphingomonas strain α449 culture.

FIG. 2 shows α449 42 L production fermentation using SEB-022-SF+MSPmedium.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, the present invention provides nucleic acidmolecules that encode a modulator of exopolysaccharide biosynthesis, andmethods of making and using the same to identify bacteria that no longerrespond to the modulator and, therefore, are capable of producing higherlevels of exopolysaccharide. Use of genetic techniques to createimproved exopolysaccharide producing bacteria is desired for the ease oflarge-scale production of biopolymers, such as sphingan polysaccharides.However, the synthesis of biopolymers is highly regulated becausebacterial survival will be compromised if an unnecessary expenditure ofmetabolic energy synthesizing such large molecules occurs at a time, forexample, when energy must be directed to growth. Moreover, theregulation of exopolysaccharide synthesis and assembly is very complexbecause a large number of proteins are required to create and exportthese macromolecules. The present invention solves these problem byidentifying and using a nucleic acid sequence that encodes a negativeregulator of sphingan biosynthesis to generate Sphingomonas derivativesthat no longer respond to the negative regulator and that, consequently,are capable of producing higher than normal amounts of sphinganexopolysaccharide.

In the present description, any concentration range, percentage range,or integer range is to be understood to include the value of any integerwithin the recited range and, when appropriate, fractions thereof (suchas one tenth and one hundredth of an integer), unless otherwiseindicated. As used herein, “about” or “comprising essentially of”mean±15%. The use of the alternative (e.g., “or”) should be understoodto mean either one, both or any combination thereof of the alternatives.When a term is provided in the singular, the inventors also contemplatethe plural of that term. In addition, it should be understood that theindividual compounds, or groups of compounds, derived from the variouscombinations of the structures and substituents described herein, aredisclosed by the present application to the same extent as if eachcompound or group of compounds was set forth individually. Thus,selection of particular structures or particular substituents is withinthe scope of the present invention.

Sphingan Polysaccharides

The term “sphingan” and the phrase “sphingan exopolysaccharide,” as usedherein, refer to a group of related, but distinct, polysaccharidessecreted by members of the genus Sphingomonas (Pollock, J. Gen.Microbiol. 139:1939-1945, 1993). Common members of the genusSphingomonas, and the sphingans they produce, include Sphingomonaspaucimobilis (ATCC 31461, formerly Pseudomonas elodea), which producesexopolysaccharide S-60 (gellan) (see, e.g., U.S. Pat. Nos. 4,377,636;4,326,053; 4,326,052 and 4,385,123); Sphingomonas sp. ATCC 21423, whichproduces exopolysaccharide S-7 (see, e.g., U.S. Pat. No. 3,960,832);Sphingomonas sp. ATCC 31554, which produces exopolysaccharide S-88 (see,e.g., U.S. Pat. Nos. 4,331,440 and 4,535,153); Sphingomonas sp. ATCC31555, which produces exopolysaccharide S-130 (welan) (see, e.g., U.S.Pat. No. 4,342,866); Sphingomonas sp. ATCC 31961, which producesexopolysaccharide S-194 (rhamsan) (see, e.g., U.S. Pat. No. 4,401,760);Sphingomonas sp. ATCC 31853, which produces exopolysaccharide S-198(see, e.g., U.S. Pat. No. 4,529,797); Sphingomonas sp. ATCC 53159, whichproduces exopolysaccharide S-657 (diutan) (see, e.g., U.S. Pat. No.5,175,278); Sphingomonas sp. ATCC 53272, which producesexopolysaccharide NW11 (see, e.g., U.S. Pat. No. 4,874,044);Sphingomonas sp. FERM BP-2015 (previously Alcaligenes latus B-16), whichproduces biopolymer B-16 (alcalan) (see, e.g., U.S. Pat. No. 5,175,279);and the like.

The structures of the sphingans are all somewhat related. The main chainof each sphingan consists of a related sequence of four sugars,including D-glucose, D-glucuronic acid, L-mannose, and L-rhamnose.Polysaccharide members of the sphingan group are distinguishable fromeach other by virtue of the carbohydrates that form the polymerbackbone, the presence or absence of acyl substituents (e.g., acetyl,glyceryl, pyruvyl, hydroxybutanoyl), and the presence or absence ofside-chains. For example, sphingan polysaccharides may containcarbohydrate side-chains, and acetyl or pyruvyl groups attached to thepolymer backbone carbohydrate. See, e.g., Mikolajczak et al., Appl. Env.Microbiol. 60:402, 1994. In certain embodiments, members of the sphinganexopolysaccharide family may be represented by the following generalrepeating chemical structure:

wherein Glc is glucose; GlcA is glucuronic acid or 2-deoxy-glucuronicacid; Rha is rhamnose; Man is mannose; X may be Rha or Man; Z isattached to Glc residue 2 and may be α-L-Rha-(1-4)-α-L-Rha, α-L-Man, orα-L-Rha; Y is attached to Glc residue 1 and may be13-D-Glc-(1-6)-α-D-Glc, 13-D-Glc-(1-6)-13-D-Glc, or α-L-Rha, subscriptsm and n may be independently from 0 to about 1, and wherein the“reducing end” of the polymer is toward the X residue of the backbone.In standard practice, the reducing end of an oligosaccharide orpolysaccharide is placed on the right. As used herein, the term“backbone” or “main chain” refers to that portion of the structure thatexcludes chains Y and Z (i.e., when m and n are equal to 0). Forexample, the main chain of gellan (S-60) comprises the sugars D-glucose,D-glucuronic acid and L-rhamnose in a 2:1:1 molar ratio, which arelinked together to form a tetrasaccharide repeat unit with the followingsubunit order: glucose, glucuronic acid, glucose, rhamnose. The mainchain of another sphingan, diutan (S-657), differs from gellan in thatit has an additional disaccharide side chain of L-rhamnose attached toglucose residue 2, which forms a hexapolysaccharide repeat unit. Stillanother sphingan, welan (S-130), has the same primary structure asgellan but with a side chain of a single L-mannose or a singleL-rhamnose attached to glucose residue 2, which forms apentapolysaccharide repeat unit.

Some members of the sphingan polysaccharide family are acetylated atvarious positions. For example, as described herein, gellan (alsoreferred to as “gellan gum”) has the same carbohydrate backbone as welan(i.e., X=Rha), lacks a side chain sugar as welan has (i.e., m=0 andn=0), and, in contrast to welan, glucose residue 1 is fully glycerylatedand partially acetylated. Gellan gum has, on average, about one glycerylgroup per tetrasaccharide repeat unit and about one acetyl group per tworepeat units. Another sphingan containing acyl groups is diutan (S-657),which contains acetyl groups at position 2 and/or position 6 of glucoseresidue 2. As is known in the art and described herein, sphinganpolysaccharides may be subjected to conditions that promote deacylationin a conventional manner to remove the acyl groups. Thus, “deacylated,”as used herein, refers to a sphingan polysaccharide that lacks one ormore acyl substituents, such as glyceryl and acetyl groups.

By way of background, Sphingomonas can produce a sphinganexopolysaccharide in the form of a capsule or in the form of slime. Asused herein, “capsule” refers to a polysaccharide attached to thesurface of a producing bacterial cell, which remains attached to thecells even after aqueous dilution, sedimentation, or centrifugation.Typically, some form of physical (e.g., heat) or chemical treatment isrequired to separate capsule exopolysaccharide from a bacterial cell. Asused herein, “slime” refers to a polysaccharide that is not attached tothe producing bacterial cell. That is, exopolysaccharide in slime formcan be substantially separated from bacterial cells by, for example,centrifugation of the fermentation broth or aqueous dilution of thebroth (even in the absence of heat treatment or other physical orchemical treatment). As is known in the art, slime formexopolysaccharide producing bacteria can be distinguished from thoseproducing capsular polysaccharide by observation with a lightmicroscope: encapsulated Sphingomonas form multicellular aggregates,while slime-forming bacteria are evenly dispersed. In addition, anencapsulated Sphingomonas can be converted into a slimy Sphingomonas by,for example, mutagenesis as described in U.S. Pat. No. 6,605,461.

The term “Sphingomonas,” as used herein, refers to a genus ofgram-negative bacteria and derivative strains thereof that produceexopolysaccharides (e.g., sphingans), as described herein. Thesphingan-producing family of gram-negative bacteria was first identifiedas belonging to the genus Sphingomonas in 1993 (see Pollock, J. Gen.Microb. 139: 1939, 1993). Sphingomonas useful in the present inventioninclude parent Sphingomonas strains and derivatives thereof. As usedherein, “parent strain” refers to bacteria or an individual bacteriumbefore any treatment, such as chemical, biological or other types ofmutagenesis, that will modify the genetic content (e.g., a substitution,insertion, or deletion within a genomic or extragenomic sequence) orphenotype of a parent strain. A person having ordinary skill in the artwill understand that a mutant Sphingomonas can be a “parent strain,”such as a mutant Sphingomonas derivative that subsequent to undergoingmutation produces exopolysaccharide in slime form rather than capsuleform. As used herein, the term “derivative,” when referring to aSphingomonas species, strain, or bacterium, means any Sphingomonasspecies, strain, or bacterium that retains essentially the same or anenhanced capability of producing a sphingan exopolysaccharide, asdescribed herein.

In addition, as used herein, “genetically mutated,” “geneticallymodified,” “mutagenized,” and “mutant” refer to the quality of havingone or more spontaneous or induced mutations, and of exhibitingproperties that distinguish the mutated bacterium or strain from theparent bacterium or strain. Thus, a mutant Sphingomonas species, strain,or bacterium that hyper-produces a sphingan exopolysaccharide iscontemplated as a derivative of the parent Sphingomonas species, strain,or bacterium for purposes of the present invention. “Inducedmutagenesis,” as used herein, means the treatment of bacterial cellswith agents commonly known to induce a genetic alteration in DNA,including chemical compounds, electromagnetic radiation, ionizingradiation, and biological agents (such as viruses, plasmids, insertionelements or transposons). In one preferred embodiment, the inventionprovides a mutated Sphingomonas that produces exopolysaccharide in thepresence of a negative regulator of exopolysaccharide biosynthesis.

The term “biosynthesis” as used herein describes the biologicalproduction or synthesis of any type of macromolecule, such as a nucleicacid, a polypeptide, or a polysaccharide (e.g., sphingans ofSphingomonas or xanthans of Xanthomonas), which may include severalbiosynthetic steps to arrive at an intermediate or final product. Forexample, sphingan exopolysaccharides are synthesized from individualcarbohydrate units in a series of steps controlled by a number ofenzymes (e.g., glycosyl transferases) of the bacteria. The term“biomass” refers to the exopolysaccharide plus bacterial cells in abacterial culture.

In certain embodiments, Sphingomonas of the present invention produceexopolysaccharide in slime form. For example, Sphingomonas strainsgenetically mutated to synthesize and export sphingan exopolysaccharidesin a slime form can be used as the “parent strain” in the context of theinstant invention. Examples of Sphingomonas parent strains that areuseful in the present invention include strain X287 (ATCC PTA-3487),which produces gellan gum (S-60) in a slime form; strain X530 (ATCCPTA-3486) and α252 (ATCC PTA-5128), which produce welan gum (S-130) in aslime form; strain Z473 (ATCC PTA-3485), which producesexopolysaccharide S-88 in a slime form; and strain X031 (ATCC PTA-3488),which produces exopolysaccharide S-7 in a slime form (see, e.g., U.S.Pat. Nos. 5,338,841 and 6,605,461). One advantage of a Sphingomonasstrain that produces sphingan exopolysaccharide in slime form is thatthe fermentation broth viscosity can be significantly reduced, whichshould allow accumulation of increased amounts of a desiredpolysaccharide. However, exemplary slime form Sphingomonas strain X287(ATCC PTA-3487) produced gellan gum at a level similar to parent strainSphingomonas paucimobilis (ATCC 31461), while still advantageouslydecreasing broth viscosity. Accordingly, one aspect of the invention isthe identification of a nucleic acid sequence capable of modulating theexpression of polysaccharides, such as sphingans.

Sphingan Biosynthesis Modulator

Many species of bacteria synthesize and secrete acidic polysaccharidesif supplied with a readily convertible carbon source, such as glucose,and an adequate amount of oxygen. Several members of the bacterial genusSphingomonas produce a variety of acidic polysaccharides, collectivelyknown as sphingans. Under certain environmental situations, bacteriawill conserve energy by minimizing exopolysaccharide production via anegative regulatory system. The present invention is directed generallyto nucleic acid sequences that encode one or more polypeptides capableof altering (e.g., increasing or decreasing in a statisticallysignificant manner) exopolysaccharide production in Sphingomonas. Thisinvention also pertains to methods of making bacteria capable ofhyper-producing an exopolysaccharide in slime form by screening forbacteria that no longer respond to the modulator of exopolysaccharidebiosynthesis. Thus, in certain preferred embodiments of the instantinvention, an isolated nucleic acid molecule that encodes one or morepolypeptides capable of modulating exopolysaccharide production inSphingomonas species is used to make mutant bacteria capable ofhyper-producing (i.e., producing, in a statistically significant manner,a greater quantity than the parent strain) an exopolysaccharide, forexample, in slime form.

Suitable nucleic acid molecules and polypeptides capable of modulatingexopolysaccharide production include, but are not limited to, naturallyoccurring nucleic acid molecules and polypeptides, and derivatives oranalogues thereof. A “purified peptide, polypeptide, or protein” or“purified nucleic acid molecule” are sequences that are individuallyessentially free from contaminating cellular components, such ascarbohydrate, lipid, nucleic acid (DNA or RNA), or other proteinaceousimpurities associated with the nucleic acid molecule or polypeptide innature. Preferably, the purified nucleic acids and polypeptides aresufficiently free of contaminants for use in the chemical couplingreactions of the instant invention or for other uses as needed. An“isolated peptide, polypeptide, or protein” or “isolated nucleic acidmolecule” are sequences that have been removed from their originalenvironment, such as being separated from some or all of the co-existingmaterials in a natural environment (e.g., a natural environment may bean unaltered cell).

Standard molecular genetic techniques can be used to identify andisolate a nucleic acid encoding a modulator of exopolysaccharidebiosynthesis. As used herein, “modulator” refers to a compound(naturally or non-naturally occurring), such as a biologicalmacromolecule (e.g., nucleic acid, protein, non-peptide or organicmolecule), that typically has activity (directly or indirectly) as aninhibitor or an activator (or both) in a biological process or processes(e.g., repressor, activator, sigma factor, antimicrobial agent,antisense molecule, interference molecule, enzyme, and the like) inassays or screens described herein. “Modulation” refers to the capacityof a compound to enhance or inhibit (or both) a functional property of abiological activity or process in a statistically significant manner(e.g., gene expression, enzyme activity, or receptor binding). Forexample, using methods well known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) edition, Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1989), a genomic expressionlibrary from Sphingomonas sp. S-88 (ATCC 31554) can be constructed,transformed into bacteria that express exopolysaccharide in capsule orslime form (e.g., Sphingomonas strain X031), preferably slime form, andscreened for recombinant bacteria that no longer expressexopolysaccharide.

Using such methods for the instant invention, a nucleic acid molecule(of approximately 2,600 nucleotides, SEQ ID NO:3) was identified andisolated from a Sphingomonas sp. S-88 genome, which encodes one or morepolypeptides that inhibit, directly or indirectly, sphinganbiosynthesis. Furthermore, a fragment of 573 nucleotides (SEQ ID NO:1)isolated from SEQ ID NO:3 was identified as sufficient to inhibitsphingan biosynthesis in Sphingomonas, including in S-7, S-88, S-130,S-194, S-198, and NW11. SEQ ID NO:1 is also capable of inhibitingxanthan biosynthesis in Xanthomonas (e.g., Xanthomonas campestris X59,ATCC 55298). In a preferred embodiment, the modulator ofexopolysaccharide biosynthesis is a nucleic acid molecule that encodes apolypeptide modulator, preferably the modulator is a negative regulatorof exopolysaccharide biosynthesis. That is, a host cell that expresses anucleic acid that encodes a negative regulator of exopolysaccharidebiosynthesis will result in inhibition of expression ofexopolysaccharide in that host cell.

By way of background and not wishing to be bound by theory, nucleic acidsequence analysis of SEQ ID NO:3 revealed that potentially threepolypeptides are encoded by this sequence, including a homologue of3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase (DAHPS), a newpolypeptide referred to as MPG, and a new polypeptide referred to asSpsN. DAHPS is involved in aromatic amino acid biosynthesis. Inaddition, SEQ ID NO:1 encodes SpsN, MPG and the carboxy-terminalterminal end of DAHPS. Thus, and as described in greater detail herein,one or more of MPG and SpsN can function as a modulator, directly orindirectly, of sphingan and xanthan biosynthesis. Example 3 describeshow the presence of an spsN nucleic acid sequence is necessary, andpotentially sufficient, to inhibit certain bacterial exopolysaccharidebiosynthesis. In a preferred embodiment, the invention provides anucleic acid molecule as set forth in SEQ ID NO:3 or SEQ ID NO:1, whichencodes at least one polypeptide capable of altering exopolysaccharideproduction in a Sphingomonas or Xanthomonas species. Furthermore, theinstant invention should be understood to also pertain to a nucleic acidmolecule as set forth in SEQ ID NO:3 or SEQ ID NO:1 that itselffunctions as a modulator or encodes another nucleic acid that functionsas a modulator. Thus, in another preferred embodiment, there is provideda nucleic acid molecule as set forth in SEQ ID NO:3 or SEQ ID NO:1 thatis capable of altering exopolysaccharide production in a Sphingomonas orXanthomonas species.

While particular embodiments of isolated nucleic acids encoding amodulator of exopolysaccharide biosynthesis are depicted in SEQ ID NOS:3and 1, within the context of the present invention, reference to one ormore isolated nucleic acids includes variants of these sequences thatare substantially similar in that they encode native or non-nativeproteins, polypeptides or peptides with similar structure and functionto a modulator of exopolysaccharide biosynthesis, such as SEQ ID NO:4 orSEQ ID NO:2. As used herein, the nucleotide sequence is deemed to be“substantially similar” if: (a) the nucleotide sequence is derived fromthe coding region of SEQ ID NO:3 or 1 isolated from a Sphingomonas orXanthomonas (including, for example, portions of the sequence or allelicvariations of the sequences discussed above) and is capable of alteringexopolysaccharide biosynthesis; (b) the nucleotide sequence is capableof hybridization to the nucleotide sequences of the present inventionunder moderate or high stringency; (c) the nucleotide sequences aredegenerate (i.e., sequences which code for the same amino acids using adifferent codon sequences) as a result of the “wobble” in the geneticcode for the nucleotide sequences defined in (a) or (b); or (d) is acomplement of any of the sequences described in (a), (b) or (c).Polynucleotide variants may contain one or more substitutions,additions, deletions, and/or insertions such that the activity of theencoded polypeptide, preferably, is not substantially diminished, asdescribed herein.

In one embodiment, preferred is an isolated nucleic acid moleculecomprising a sequence that remains hybridized under highly stringentconditions to a probe, wherein the probe consists of SEQ ID NO:1 or acomplement of SEQ ID NO:1. In a preferred embodiment, the presentinvention provides an isolated nucleic acid molecule that consists ofSEQ ID NO:1 or a complement of SEQ ID NO:1. Certain isolated nucleicacids of the inventions are, therefore, useful for detecting thepresence of a nucleic acid that encodes a modulator of exopolysaccharidebiosynthesis or an analogue, homologue, or derivative thereof, or areuseful for expressing a polypeptide capable of alteringexopolysaccharide biosynthesis. As used herein, “nucleic acid molecule”or “polynucleotide” refers to a polymeric form of nucleotides of a leastten bases in length, either ribonucleotides or deoxynucleotides or amodified from of either type of nucleotide. The term includes single anddouble stranded forms of DNA or RNA or any of a number of known naturaland non-natural chemical variants of nucleic acids, for instance,nucleic acids having greater resistance to degradation, such as thosecontaining phosphorothioates. In certain embodiments, the nucleic acidmolecules that encode a modulator of exopolysaccharide biosynthesis areDNA or RNA.

“Moderately stringent hybridization conditions” and “highly stringenthybridization conditions” are conditions of hybridization of a probenucleotide sequence to a target nucleotide sequence according toestablished principles of nucleotide base-pairing and hydrogen bondformation wherein hybridization will only be readily detectable when aportion of the target sequence is substantially similar to thecomplement of the probe sequence. Hybridization conditions vary withprobe size as well as with temperature, time, and salt concentration ina manner known to those having ordinary skill in the art. For example,moderate hybridization conditions for a 50 nucleotide probe wouldinclude hybridization overnight in a buffer containing 5×SSPE(1×SSPE=180 mM sodium chloride, 10 mM sodium phosphate, 1 mM EDTA (pH7.7), 5×Denhardt's solution (100×Denhardt′ s=2% (w/v) bovine serumalbumin, 2% (w/v) Ficoll, 2% (w/v) polyvinylpyrrolidone) and 0.5% SDSincubated overnight at 55-60° C. Post-hybridization washes at moderatestringency are typically performed in 0.5×SSC (1×SSC=150 mM sodiumchloride, 15 mM trisodium citrate) or in 0.5×SSPE at 55-60° C. Highlystringent hybridization conditions typically would include 2×SSPEovernight at 42° C., in the presence of 50% formamide, followed by oneor more washes in about 0.1×SSC to about 0.2×SSC, and 0.1% SDS at 65° C.for 30 minutes or more.

The isolated nucleic acids encoding a modulator of exopolysaccharidebiosynthesis according to this invention can be obtained using a varietyof methods. For example, as described above, a nucleic acid molecule maybe obtained from a cDNA or genomic expression library by screening withan antibody or antibodies reactive with an SpsN or MPG polypeptide (see,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, 1989; Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing, 1987). Further, random-primed PCR can beemployed (see, e.g., Methods in Enzymol. 254:275, 1995). In addition,variations of random-primed PCR can also be used, especially when aparticular gene or gene family is desired. In one such method, one ofthe primers is a random primer and the other is a degenerate primerbased on the amino acid sequence or nucleotide sequence encoding amodulator of exopolysaccharide biosynthesis.

Other methods may also be used to obtain isolated nucleic acid moleculesthat encode a modulator of exopolysaccharide biosynthesis. For example,a nucleic acid molecule can be isolated by using the sequenceinformation provided herein to synthesize a probe that can be labeled,such as with a radioactive label, enzymatic label, protein label,fluorescent label, or the like, and hybridized to a genomic library or acDNA library constructed in, for example, a phage, plasmid, phagemid, orviral vector designed for replication or expression in one or moreselected host cells (see, e.g., Sambrook et al., supra; Ausubel et al.,supra). DNA representing RNA or genomic nucleic acid sequence can alsobe obtained by amplification using sets of primers complementary to 5′and 3′ sequences of the isolated nucleic acid sequences provided in SEQID NOS:1 and 3, or to variants thereof, as described above. For ease ofcloning, restriction enzyme sites can also be incorporated into theprimers. Thus, the present invention includes nucleic acid moleculesthat are useful as primers for use in PCR amplification proceduresspecific for the amplification of at least one mRNA or DNA encoding amodulator of exopolysaccharide biosynthesis, particularly in samplesderived from Sphingomonas or Xanthomonas (for PCR procedures see, e.g.,U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,965,188; and Innis et al., PCRStrategies, Academic Press, San Diego, 1995). Such PCR amplificationmethods are known in the art and include primer extension PCR, real timePCR, reverse transcriptase PCR (Freeman et al., BioTechniques 26:112,1999), inverse PCR (Triglia et al., Nucleic Acids Res. 16:8186, 1988),capture PCR (Lagerstrom et al., PCR Methods Applic. 1:111, 1991),differential primer extensions (WO 96/30545), and other PCRamplification methods known in the art or later developed (see, e.g.,Innis et al., PCR Strategies, Academic Press, San Diego. 1995).

In operation, PCR methods generally include the use of primer moleculesthat are chemically synthesized, but they may be generated enzymaticallyor produced recombinantly. PCR primers generally comprise two nucleotidesequences, one with sense orientation (5′->3′) and one with antisenseorientation (3′->5′), employed under preferred conditions foridentification of a specific gene or condition. The same PCR primers,nested sets of oligomers or a degenerate pool of oligomers can beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences. These nucleic acidmolecules can also be used individually or in combination as probes toidentify contactin mRNA or DNA molecules in a sample. These nucleic acidmolecules include:

5′-GACGGATCCTTGCCGAGGTGCG-3′, (SEQ ID NO: 5)5′-CGACGGCCACTACTAGCGTTCGAACG-3′, (SEQ ID NO: 6)5′-GTCCGTCGGTATCTACGGCTTCGAACG-3′, (SEQ ID NO: 7)SEQ ID NO: 5 is a forward primer, while SEQ ID NO:6 and SEQ ID NO:7 arereverse primers.

The nucleic acid molecules of the present invention can be made by avariety of methods known in the art. For example, nucleic acid moleculescan be made using synthetic procedures or molecular biology techniquesknown in the art (see, e.g., Sambrook et al., supra). The length of thenucleic acid molecules of the present invention can be readily chosen byone skilled in the art depending on the particular purpose that thenucleic acid molecule is to be used for. For PCR primers, the length ofthe nucleic acid molecule is preferably between about 10 nucleotides andabout 50 nucleotides in length, more preferably between about 12nucleotides and about 30 nucleotides in length, and most preferablybetween about 15 nucleotides and about 25 nucleotides in length. Forprobes, the length of the nucleic acid molecule is preferably betweenabout 20 nucleotides and about 1,000 nucleotides in length, morepreferably between about 100 nucleotides in length and about 500nucleotides in length, and most preferably between about 150 nucleotidesand about 400 nucleotides in length.

Variants (including alleles) of the isolated SEQ ID NOS:1 and 3 nucleicacid sequences provided herein can be readily obtained from naturalvariants (e.g., polymorphisms, mutants and other serotypes) eithersynthesized or constructed. Many methods have been developed forgenerating mutants (see, generally, Sambrook et al., supra; Ausubel etal., supra). Briefly, preferred methods for generating nucleotidesubstitutions utilize an oligonucleotide that spans the base or bases tobe mutated and contains the mutated base or bases. The oligonucleotideis hybridized to complementary single stranded nucleic acid and secondstrand synthesis is primed from the oligonucleotide. The double-strandednucleic acid is prepared for transformation into host cells, such as E.coli or other prokaryotes, and yeast or other eukaryotes. Standardscreening and vector amplification protocols are used to identify mutantsequences and obtain high yields.

Similarly, deletions or insertions of a nucleic acid molecule thatencodes a modulator of exopolysaccharide biosynthesis may be constructedby any of a variety of known methods. For example, the sequence may bedigested with restriction enzymes or nucleases and be religated suchthat sequences are deleted, added, or substituted. Similarly, a varietyof transposons and other insertional elements may be used to makerecombinants having deletions and insertions. Thus, in one example, anspsN mutant containing a Tn10Kan transposon in the spsN coding sequence,as described herein, can be made according to methodologies known in theart. Other means of generating variant sequences, also known in the art,may be employed without requiring undue experimentation (for examplessee Sambrook et al., supra, and Ausubel et al., supra). Moreover,verification of variant sequences is typically accomplished byrestriction enzyme mapping, sequence analysis, hybridization, and thelike. Variants that encode a modulator of exopolysaccharide biosynthesisthat are capable of altering exopolysaccharide biosynthesis areparticularly useful in the context of this invention.

A person having ordinary skill in the art would appreciate that otherspsN encoding sequences can be identified and isolated in a similarmanner. For example, analogues or derivatives thereof of spsN, or spsNhomologues from other species of Sphingomonas or other related bacteria(such as Xanthomonas or Pseudomonas) can be identified and isolatedusing assays described herein. Thus, in certain embodiments, preferredmodulators of exopolysaccharide biosynthesis in Sphingomonas orXanthomonas comprise a nucleic acid sequence as set forth in SEQ ID NO:3or SEQ ID NO:1, and analogues or derivatives thereof of SEQ ID NO:3 orSEQ ID NO:1 that are capable of altering exopolysaccharide biosynthesis.As used herein, the terms “derivative” and “analogue” when referring toa modulator of exopolysaccharide biosynthesis, refer to any modulator ofexopolysaccharide biosynthesis that retains essentially the same (atleast 50%, and preferably greater than 70%, 80%, 90%, or 95%) orenhanced biological function or activity as such parent modulator, asnoted above. The biological function or activity of such analogues andderivatives can be determined using standard methods (e.g., plate assay,gel analysis, transcription assay, translation assay), such as with theassays described herein and known in the art. For example, an analogueor derivative may be a proprotein that can be activated by cleavage, ormay be a precursor that can be activated or stabilized by an amino acidmodification, to produce an active modulator of exopolysaccharidebiosynthesis. Alternatively, a modulator of exopolysaccharidebiosynthesis and analogues or derivatives thereof can be identified bytheir ability to specifically bind to one or more anti-modulatorantibodies.

Another example of an analogue or derivative includes a modulator ofexopolysaccharide biosynthesis (e.g., SpsN) that has one or moreconservative amino acid substitutions, as compared with the amino acidsequence of a naturally occurring modulator. Among the common aminoacids, a “conservative amino acid substitution” is illustrated, forexample, by a substitution among amino acids within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine, or a combination thereof. Furthermore,an analogue or derivative of a modulator may include, for example,non-protein amino acids, such as precursors of normal amino acids (e.g.,homoserine and diaminopimelate), intermediates in catabolic pathways(e.g., pipecolic acid and D-enantiomers of normal amino acids), andamino acid analogues (e.g., azetidine-2-carboxylic acid, homoproline,and canavanine).

Yet other embodiments of analogues or derivatives include a modulator ofexopolysaccharide biosynthesis that retains at least about 60% identitywith the parent molecule (i.e., the “parent” molecule will depend on thestarting point, whether the parent is, for example, wild-type SpsN or ananalogue of SpsN), more preferably at least about 70%, 80%, 90%, andmost preferably at least about 95%. As used herein, “percent identity”or “% identity” is the percentage value returned by comparing the wholeof the subject polypeptide, peptide, or analogue or variant thereofsequence to a test sequence using a computer implemented algorithm,typically with default parameters. Sequence comparisons can be performedusing any standard software program, such as those provided in theLasergene® bioinformatics computing suite, which is produced by DNASTAR®(Madison, Wis.). References for algorithms, such as BLAST® or ALIGN, maybe found in, for example, Altschul, J. Mol. Biol. 219:555-565, 1991; orHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992;and preferably BLAST® is used (as used herein, BLAST® refers to one ormore of the following search algorithms: BLASTn, MEGABLAST, BLASTp,PSI-BLAST, PHI-BLAST, BLASTx, tBLASTn, tBLASTx, RPS-BLAST, CDART,VecScreen, trace BLAST, and the like), which is available at theNational Center for Biotechnology Information (NCBI, Bethesda, Md.)website (www.ncbi.nlm.nih.gov/BLAST) and results compared to databases,such as GenBank®. Other methods for comparing multiple nucleotide oramino acid sequences by determining optimal alignment are well known tothose of skill in the art (see, e.g., Peruski and Peruski, The Internetand the New Biology: Tools for Genomic and Molecular Research (ASMPress, Inc. 1997); Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997); and Bishop (ed.),Guide to Human Genome Computing, 2^(nd) Edition, Academic Press, Inc.,1998).

As used herein, “similarity” between two or more peptides orpolypeptides is generally determined by comparing the amino acidsequence of one peptide or polypeptide with one or more other peptidesor polypeptides having conserved amino acid substitutions thereto.Further, as is known in the art, a consensus sequence may be determinedfor a group of homologues, analogues, or derivatives based on the aminoacid sequence of a parent compound, such as MPG (SEQ ID NO:4) or SpsN(SEQ ID NO:2). In a preferred embodiment, the polypeptide modulatorcomprises the amino acid sequence of SEQ ID NO:2, and in an even morepreferred embodiment the modulator consists of the amino acid sequenceof SEQ ID NO:2.

An analogue or derivative may also be a fusion protein of a modulator ofexopolysaccharide biosynthesis. Fusion proteins, or chimeras, includefusions of one or more modulators of exopolysaccharide biosynthesis withnon-modulator peptides or polypeptides, such as a polypeptide tag (e.g.,an epitope tag or 6×His tag), carrier, or enzyme. The peptides may alsohave a detectable label or “tag” (i.e., be labeled), such as with aradioactive label, a fluorescent label, a mass spectrometry tag, biotin,and the like.

Peptides may be produced by recombinant techniques and a variety of hostsystems are suitable for production of modulators of exopolysaccharidebiosynthesis and analogues or derivatives thereof, including bacteria(e.g., E. coli), yeast (e.g., Saccharomyces cerevisiae), insect (e.g.,Sf9), and mammalian cells (e.g., CHO, COS-7). Many expression vectorshave been developed and are available for each of these hosts. In apreferred embodiment, vectors that are functional (i.e., capable ofreplicating) in bacteria are used in this invention, even morepreferably the vectors are broad host range plasmids, such as pRK311(Ditta et al., Plasmid 13:149, 1985) and pMMB(EH) (Fürste et al., Gene48:119, 1986). However, at times, it may be preferable to have vectorsthat are functional in other hosts or more than one host. Vectors andprocedures for cloning and expression in E. coli are discussed hereinand, for example, in Sambrook et al. (1987) and in Ausubel et al.(1995).

“Vector” refers to a nucleic acid assembly that is capable of directingthe expression of a desired polypeptide. The vector may include anexpression control sequence (e.g., transcriptional promoter/enhancerelements) that is operably linked to the nucleic acid coding sequence orisolated nucleic acid molecule(s) of interest. The vector may becomposed of DNA, RNA, or a combination of the two (e.g., a DNA-RNAchimera). Optionally, the vector may include a polyadenylation sequence,one or more restriction sites, as well as one or more selectablemarkers, such as neomycin phosphotransferase or hygromycinphosphotransferase, as needed. Additionally, depending on the host cellchosen and the vector employed, other genetic elements such as an originof replication, additional nucleic acid restriction sites, enhancers,sequences conferring inducibility or repressibility of transcription,and selectable markers, may also be incorporated into the vectorsdescribed herein.

“Cloning vector” refers to nucleic acid molecules, such as a plasmid,cosmid, or bacteriophage, which are capable of replicating autonomouslyin a host cell. Cloning vectors typically contain one or a small numberof restriction endonuclease recognition sites, at which foreignnucleotide sequences can be inserted in a determinable fashion withoutloss of an essential biological function of the vector. Cloning vectorsalso typically contain a marker gene (e.g., antibiotic resistanceencoding gene) that is suitable for use in the identification andselection of cells transformed with the cloning vector. Marker genestypically encode proteins that provide resistance to antibiotics, suchas tetracycline, kanamycin, ampicillin, and the like.

As used herein, “nucleic acid expression construct” refers to a nucleicacid molecule construct containing a nucleic acid sequence that isexpressed in a host cell. Typically, expression of a nucleic acidsequence of interest is placed under the control of an expressioncontrol sequence (e.g., promoter), and optionally, under the control ofat least one regulatory element. Such an expressed sequence is said tobe “operably linked to” the promoter. Similarly, a regulatory elementand a promoter are operably linked if the regulatory element alters(i.e., increases or decreases with statistical significance) theactivity of the promoter. As used herein, “expression control sequence”refers to a nucleotide sequence that directs the transcription of astructural gene. Typically, an expression control sequence is located inthe 5′ region of a gene, proximal to the transcriptional start site of astructural gene. If an expression control sequence is an induciblepromoter, then the rate of transcription may, for example, be increasedby the addition of an inducing agent or decreased by the addition of aninhibiting agent. In contrast, an inducing or an inhibiting agent doesnot affect the rate of transcription of a constitutive promoter.

A person having ordinary skill in the art is capable of selecting asuitable expression control sequence and suitable host for expressing,for example, an isolated nucleic acid sequence encoding a peptide havingthe amino acid sequence of SEQ ID NO:4 or variants thereof, wherein thevariants comprise amino acid sequences having conservative amino acidsubstitutions or having at least 80% sequence identity to SEQ ID NO:4,and wherein the variants are capable of altering exopolysaccharidebiosynthesis. In a preferred embodiment, there is provided a recombinantexpression vector comprising at least one promoter operably linked to anucleic acid molecule as set forth in SEQ ID NO:1 or 3, or a variantthereof, that encodes at least one polypeptide capable of alteringexopolysaccharide biosynthesis in Sphingomonas.

A DNA sequence encoding a modulator of exopolysaccharide biosynthesismay be introduced into an expression vector appropriate for a particularhost. In certain embodiments, the nucleic acid sequence may be clonedinto a vector or expression vector to generate a fusion protein. Thefusion partner may be chosen to be a polypeptide tag, such thatisolation of a fusion protein is facilitated. The fusion carrier mayprevent modulator degradation by host proteases, or the fusion partnermay further function to transport the fusion peptide to inclusionbodies, the periplasm, the outer membrane, or the extracellularenvironment. Thus, the instant invention contemplates a recombinantexpression vector comprising at least one promoter operably linked to anucleic acid molecule as set forth in SEQ ID NO:1 or 3 or a variantthereof that encodes at least one polypeptide, which is expressed as afusion protein capable of altering exopolysaccharide biosynthesis inSphingomonas. Typically, the protein portion fused to a modulator ofexopolysaccharide biosynthesis will be encoded by a second nucleic acidsequence; preferably the second nucleic acid sequence encodes apolypeptide tag or an enzyme, as described herein and is known in theart. In certain embodiments, the expression vector promoter willpreferably be regulated and, in certain circumstances, the vector willpreferably be a plasmid that replicates in Sphingomonas, Pseudomonas, orXanthomonas. In a preferred embodiment, the plasmid is pRK311 containingSEQ ID NO:1 (X029, ATCC PTA-5127) or SEQ ID NO:3.

As used herein, “host cell” refers to any prokaryotic or eukaryotic cellthat contains either a cloning vector or a nucleic acid expressionconstruct. This term also includes those prokaryotic or eukaryotic cellsthat have been recombinantly engineered to contain cloned nucleic acidsequence(s) in the chromosome or genome of the host cell. Preferably, ahost cell is a prokaryotic cell, more preferably a bacterium, and evenmore preferably a Sphingomonas, Xanthomonas, or Pseudomonas.Sphingomonas being most preferred. In certain preferred embodiments, thehost cell is a sphingan producing bacterium, such as Sphingomonas sp.that produce gellan, welan, rhamsan, diutan, alcalan, S-7, S-88, S-198,and NW11. In one embodiment, the present invention provides a host cellSphingomonas, such as α027, containing plasmid X029 (ATCC PTA-5127). Inanother embodiment, the host cell is Xanthomonas.

Enhanced Exopolysaccharide Biosynthesis

According to another aspect of the present invention there are providedmethods for identifying and isolating bacteria that are capable ofover-producing exopolysaccharide. In particular, a modulator ofexopolysaccharide biosynthesis can be used to identifyexopolysaccharide-producing bacteria that no longer respond to themodulator of exopolysaccharide biosynthesis. For example, colonies ofbacteria that produce exopolysaccharide will have a mucoid appearance onagar plates, while non-producers will have a non-mucoid appearance.Introduction of a recombinant expression vector that encodes a modulatorof exopolysaccharide biosynthesis into an exopolysaccharide producingbacterium will suppress exopolysaccharide expression, which means thebacteria will have a non-mucoid appearance on agar plates. Thesesuppressed strains can be screened for spontaneous mutants or inducedmutants that are capable of expressing exopolysaccharide even in thepresence of a modulator of exopolysaccharide biosynthesis.

Within certain embodiments, there is provided a method for makingbacteria capable of hyper-producing an exopolysaccharide in slime form,comprising contacting bacteria suppressed for production of anexopolysaccharide in slime form with a mutagen, wherein the bacteria (i)contain a recombinant expression vector comprising at least one promoteroperably linked to a nucleic acid molecule that encodes at least onepolypeptide capable of altering exopolysaccharide production, and (ii)express a polypeptide of part (i) such that exopolysaccharide productionis suppressed; and then identifying therefrom bacteria capable ofhyper-producing exopolysaccharide in slime form in the presence of apolypeptide capable of suppressing exopolysaccharide production.

The term “hyper-producing” refers to the capability of a first bacterialstrain that produce statistically significantly more exopolysaccharidethan a second strain from which the first bacterial strain was directlyor indirectly derived. A first bacterial strain is “directly derived”from a second bacterial strain if the first strain was obtained from asingle cycle of mutagenesis and/or selection of the second strain. Afirst bacterial strain is “indirectly derived” from a second bacterialstrain if the first strain was obtained from multiple cycles ofmutagenesis and/or selection of the second strain.

In one preferred embodiment, there is provided a method for makingbacteria that are capable of hyper-producing an exopolysaccharide inslime form, comprising (A) contacting (a) a mutagen with (b) bacteriathat are capable of producing an exopolysaccharide in slime form,wherein the bacteria (i) contain a recombinant expression vectorcomprising at least one promoter operably linked to a nucleic acidmolecule that encodes at least one polypeptide, wherein the nucleic acidmolecule encodes at least one polypeptide capable of alteringexopolysaccharide production, and (ii) express said at least onepolypeptide of (i) such that exopolysaccharide production is suppressed,under conditions and for a time sufficient to produce mutagenizedbacteria; and (B) identifying among said mutagenized bacteria one or aplurality of bacteria that are capable of hyper-producingexopolysaccharide in slime form. In certain preferred embodiments, thebacteria are Sphingomonas, such as S-60, S-7, S130, S-7, S-194, S-198,and NW11, and the mutagen is, for example, ethylmethane sulfonate (EMS),N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or another suitable mutagenas known in the art. In another preferred embodiment, the modulator ofexopolysaccharide biosynthesis is encoded by SEQ ID NO:1 or 3, andpreferably by a nucleic acid molecule comprising a sequence that encodesthe amino acid sequence of SEQ ID NO:2 or 4. In one particularlypreferred embodiment, the parent bacterium is Sphingomonas α027, and themutant of α027 that no longer responds to a modulator ofexopolysaccharide biosynthesis is Sphingomonas α062 (ATCC PTA-4426) (formore details, see Examples). In another embodiment, the parent bacteriumis Xanthomonas campestris X59, and the mutant of X59 that no longerresponds to a modulator of exopolysaccharide biosynthesis is Xanthomonascampestris α449 (ATCC PTA-5064). Preferably, the mutant strain produces(in a statistically significant manner) more exopolysaccharide than theparent strain, such as at least about 10% to about 20% more, preferablyat least about 20% to about 30% more, and more preferably at least about30% to about 40% more.

Production of Exopolysaccharides

Another aspect of the present invention relates to the enhancedproduction of exopolysaccharide. For example, to produce sphinganexopolysaccharide, genetically mutated Sphingomonas bacteria arecultured under suitable fermentation conditions, which are well known inthe art and which are generally described in U.S. Pat. No. 5,854,034.Briefly, a suitable medium for culturing the genetically mutatedSphingomonas is an aqueous medium that generally contains a source ofcarbon such as, for example, carbohydrates including glucose, lactose,sucrose, maltose or maltodextrins; a nitrogen source such as, forexample, inorganic ammonium, inorganic nitrate, organic amino acids orproteinaceous materials such as hydrolyzed yeast, soy flour or casein;distiller's solubles or corn steep liquor; inorganic salts and vitamins.A wide variety of fermentation media will support the bacterialproduction of sphingans according to the present invention. Thecarbohydrates are included in the fermentation broth in varying amounts,but usually between about 1% and about 5% by weight of the fermentationmedium. The carbohydrates may be added all at once prior to fermentationor alternatively, during fermentation. The amount of nitrogen may rangefrom about 0.01% to about 0.2% by weight of the aqueous medium. A singlecarbon source or nitrogen source may be used, as well as mixtures ofthese sources. Among the inorganic salts which find use in fermentingSphingomonas are salts that contain sodium, potassium, ammonium,nitrate, calcium, phosphate, sulfate, chloride, carbonate and similarions. Trace metals, such as magnesium, manganese, cobalt, iron, zinc,copper, molybdenum, iodide and borate, may also be advantageouslyincluded. Vitamins, such as biotin, folate, lipoate, niacinamide,pantothenate, pyridoxine, riboflavin, thiamin and vitamin B.sub.12 andmixtures thereof, may also be advantageously employed.

Generally, the fermentation can be carried out at temperatures between(and including) about 25° C. and 35° C., with optimum productivityobtained within a temperature range of about (and including) 28° C. to32° C. The inoculum is prepared by standard methods of volume scale-up,including shaken flask cultures and small-scale submerged stirredfermentation. The medium for preparing the inoculum can be the same asthe production medium or can be any one of several standard mediawell-known in the art, such as Luria broth or YM medium. Theconcentration of carbohydrate can be reduced in the seed cultures toless than about 1% by weight. More than one seed stage may be used toobtain the desired volume for inoculation. Typical inoculation volumesrange from about 0.5% to about 10% of the total final fermentationvolume. The fermentation vessel typically contains an agitator to stirthe contents. The vessel may also have automatic pH and foamingcontrols. The production medium is added to the vessel and sterilized inplace by heating. Alternatively, the carbohydrate or carbon source maybe sterilized separately before addition. A previously grown seedculture is added to the cooled medium (generally, at the fermentationtemperature of about 28° C. to about 32° C.) and the stirred culture isfermented for about 48 hours to about 96 hours, producing a broth havinga viscosity of from about 15,000 centipoise (cp) to about 20,000 cp, andfrom about 10 to about 15 g/L sphingan exopolysaccharide in slime form.The fermentation of a corresponding parent Sphingomonas strain willtypically provide a broth having a viscosity of from about 25,000 cp toabout 50,000 cp.

In this aspect, the invention provides an exopolysaccharide in slimeform obtained from Sphingomonas or Xanthomonas grown in submerged,stirred and aerated liquid culture. The concentration of dissolvedoxygen in the liquid culture preferably exceeds about 5% of saturationof water after 24 hours of culturing. Similar fermentations withencapsulated strains resulted in 0% dissolved oxygen after 24 hours. Thelower viscosity provided by the exopolysaccharide in slime form resultsin improved aeration which allows Sphingomonas to be productive inculture for a longer period of time. In another aspect of the presentinvention, fermentation may be carried out in a semi-batch process wherebacteria from one fermentation are used as an inoculum for a subsequentfermentation. In this aspect, for example, Sphingomonas that have beenseparated from the exopolysaccharide which they produced may be added toa fresh fermentation broth, or a fresh fermentation broth may be addedto the remaining Sphingomonas. Hence, this aspect of the inventionprecludes the need to provide a separate seed culture.

Recovery of exopolysaccharides, regardless of the conditions used toproduce them, preferably involves a precipitation step. The precipitatedexopolysaccharide may then recovered by centrifugation. A typical methodfor recovering gellan and welan gums is a follows. Immediately afterfermentation, the culture broths are heated to at least 90° C. to killthe living bacteria. The exopolysaccharides are then separated from theculture broth by precipitation with approximately 2 volumes (i.e.,culture volume equivalents) of isopropyl alcohol, and the precipitatedpolysaccharide fibers are collected, pressed, dried, and milled. Thealcohol is removed by distillation. In this most simple process, thepolysaccharide remains attached to the cells, such that when the driedand milled polysaccharide is resuspended in water, the solution is nottransparent. In the case of gellan gum, additional steps can beintroduced to purify the polysaccharide away from the bacterial cells sothat the resuspended product is more transparent. Before the alcoholprecipitation, the culture broth is centrifuged and/or filtered or bothwhile the temperature is maintained above the critical transitiontemperature between a highly viscous state and a liquefied state that isamenable to centrifugation or filtration. These processes for differentsphingans are disclosed in, for example, U.S. Pat. No. 4,326,052(gellan); U.S. Pat. No. 4,326,053 (gellan); U.S. Pat. No. 4,342,866(welan); U.S. Pat. No. 3,960,832 (S-7); and U.S. Pat. No. 4,535,153(S-88).

In certain embodiments, the present invention provides a method forhyper-producing exopolysaccharide, comprising culturing bacteria underconditions and for a time sufficient to permit exopolysaccharideproduction, wherein the bacteria hyper-produce exopolysaccharide inslime form when expressing at least one polypeptide encoded by a nucleicacid molecule that is capable of altering exopolysaccharide production;and separating the exopolysaccharide in slime form from the culture.Preferably, the bacteria are Sphingomonas, such as those that producegellan, welan, rhamsan, diutan, alcalan, S7, S88, S198, or NW11, morepreferably Sphingomonas strains α062 (ATCC PTA-4426), α063, α065 andα069. In a preferred embodiment, the culturing is by fermentation andthe bacteria produce from about 5 grams to about 80 grams ofexopolysaccharide per liter of culture, more preferably from about 10grams to about 70 grams, and even more preferably from about 20 grams toabout 60 grams. Optimal conditions for the fermentation is from about 48hours to about 96 hours at a temperature ranging from about 25° C. toabout 35° C. Preferably, the exopolysaccharide is separated by alcoholprecipitation, wherein about 1 to about 1.5 volumes of alcohol ispreferably added to the culture. Preferably, the fermentation culturehas a final viscosity ranging from about 5,000 cp to about 40,000 cp,more preferably from about 10,000 cp to about 35,000 cp, and mostpreferably from about 15,000 cp to about 30,000 cp.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, as if set forth herein, in their entirety.

EXAMPLES Example 1 Preparation of White, High Glucose ResistantSphingomonas

Sphingomonas X287 (ATCC PTA-3487) was used to inoculate 50 ml ¼YM−,having either 1% or 5% glucose in 125 ml baffled flask at 30° C. for twoweeks without shaking. No exopolysaccharide-negative mutants appearedwhen the culture was spread onto a ¼YM+ plate after such an extendedincubation. The same culture was spread onto a plate of ¼YM having 10%Glucose. Isolated colonies (83) were selected and used to inoculate 2 ml¼YM having 3% Glucose, and then incubated for 40 hrs at 30° C. withshaking at 300 rpm. These cultures were spread on high glucose plates,and 12 colonies chosen and saved as X729-X741. Six out of those twelvemutants were visually white mutants.

Colonies were used to inoculate 2 ml YM− in small tubes and shaken at300 rpm for 24 hrs at 30° C. (referred to as seed-1). Then 100 μl ofthese cultures were used to inoculate 5 ml B10G3, which culture wasplaced at an angle and incubated at 300 rpm for 24 hrs (referred to asseed-2). Then 5 ml of seed-2 was added to 20 ml B10G3 into 125 mlbaffled flask and incubated with shaking at 200 rpm during the first 17hrs, and then 400 rpm up to 46 hrs. 10 g of the final broth wasprecipitated by 20 volumes isopropyl alcohol (IPA) at room temperature.The sample was dried at 60° C. under vacuum for 2 hrs.

High Glucose Resistant, White Mutants

DW (g/L) Final pH OD₆₀₀ (Ave = 14.9 g/L) X733white 5.82 17.7 15.1X734white 5.77 17.9 15.5 X735white 5.78 17.5 15.3 X736white 5.84 16.114.7 X737white 5.55 12.5 13.6 X738white 4.31 6.0 3.6 X743white 5.78 17.015.3 X753 5.82 14.1 13.0

Parent Gellan Slime

DW (g/L) Final pH OD₆₀₀ (Ave = 12.7 g/L) X287-1 5.82 14.5 12.6 X287-25.78 14.2 12.8 X287-3 5.77 13.6 12.5 X287-4 5.78 14.2 12.8

The strain X733 inoculated in 125 ml baffled flask and incubated for 48hrs. The culture broth spread on ¼YM+ plate generated slimy-capsule formcolonies and a few yellow color colonies. Color indicator shows a hintto predict the mutation stability in some extent. One of the white slimycolonies was selected and saved as X996.

The X996 was cultivated in 100 ml B10 containing 3% and 6% Glucose using500 ml baffled flask at 160 rpm for 24 hrs with OA % inoculation.

24 hrs pH/OD600/Living cell number B10 3% Glucose 6.30/9.95/2.4 × 10¹⁰B10 6% Glucose 6.32/7.88/1.7 × 10¹⁰

It was hard to cultivate this X996 mutant reproducibly when theinoculation rate was low. This mutant did not grow well when itincubated under high rate of agitation.

This X996 was incubated for 30 hrs in 30 ml B10 medium supplemented by7% Glucose using 125 ml baffled flask.

Low rate of inoculation suppressed the X996 cell growth. This culturemedium saved at room temperature for a week without shaking. Then theculture was spread onto ¼YM 10% glucose plate. The Sphingomonasα016-α018 were isolated on high glucose containing ¼YM plates.

Example 2 Identification of Sphingomonas no Longer Responsive to SpsN

This example describes identification of a DNA sequence that suppressesbiosynthesis of sphingan and xanthan by the following steps: (1) makingan expression library of Sphingomonas S-88 genomic DNA; (2) transformingthe expression library into Sphingomonas S-7; and (3) screening forsuppression of exopolysaccharide S-7 production in Sphingomonas S-7(i.e., spread bacterial cultures on plates and observe colonies fornon-mucoid appearance). Primers SEQ ID NO:5 (22mer), and SEQ ID NOS:6and 7 (26 and 27mer, respectively) were made and used with template Z964(pBluescriptKS-BH, 716 bp, which is a fragment from Z939 (pRK311-s88nc2,2.7 Kb)) to produce a 573 base pair fragment (SEQ ID NO:1) encoding,inter alia, functional spsN. The 573 base pair PCR fragment (SEQ IDNO:1) was cloned into plasmid pRK311, and two independent clones (X026and X029) were further tested as follows:

Strain Plasmid S-7 S-88 NW11 S-130 S-194 S-198 NW11 X59X025 + + + + + + + + X026 − − − − − − − − X028 + + + + + + + + X029 − −− − − − − − Z959-1 + + + + + + + + Z959-2 + + + + + + + +Z959-3 + + + + + + + + Z967 − − − − − − − Z939 − − − − − − − − X025 =pRK311-MPG (385 bp, 22mer-26mer); X026 = pRK311-MPG + SpsN (573 bp,22mer-27mer); X028 = pRK311-MPG (385 bp, 22mer-26mer); X029 =pRK311-MPG + SpsN (573 bp, 22mer-27mer); Z959-1 to -3 = independentclones having pRK311-s88nc2 EH2700bp spsN::Tn10Kn; Z967 =pRK311-BH716bp; + means the transconjugants produce exopolysaccharide,and − means transconjugants do not produce exopolysaccharide on aselection plate.

These results demonstrate that the nucleic acid molecules encoding SpsN(e.g., X029) were capable of inhibiting polymer synthesis inSphingomonas S-7, S-130, S-194, S-198, S-88, NW11, and Xanthomonas X59.

From analysis of the sequenced amplicon, spsN (modulator ofexopolysaccharide biosynthesis) was located downstream of DAHPS(3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase). DAHPSregulates aromatic amino acid biosynthesis. The active PCR product wascloned into pRK311 and called X029 (pRK311-spsN, Tet^(r)). Thisexpression vector, which contains SEQ ID NO:1, was trans-conjugated intoSphingomonas α016, and named α027, which had a suppressed polymerproduction phenotype. Then this α027 was treated with EMS to findmutants that showed an exopolysaccharide-positive phenotype under drugpressure Tet (i.e., in the presence of X029, which encodes the modulatorof exopolysaccharide biosynthesis, SpsN). Under these conditions, someof the exopolysaccharide-positive phenotype could be due to mutations ineither SpsN or elsewhere. After selection under no drug condition to getrid of plasmid X029 (i.e., encoding SpsN), strains α061-α076 wereselected and saved for further testing (e.g., biomass production). Thesestrains were cultivated in grass tube that contain 4 ml B10 medium with5% glucose for 24 hrs. Then 3 ml of those cultures were inoculated into25 ml B10 seed medium with 3% glucose in 125 ml baffled flask at 300 rpmfor 48 hrs. From the each flask, 10 g of culture broth was precipitatedby 2 vol of IPA. Precipitated materials were dried at 60° C. for 6 hrs.

Dry Weight α061 13.18 g/L α062 13.94 somewhat capsule-slime α063 14.16slimy on plate α064 11.85 α065 15.77 slimy on plate α066 12.13 α06713.47 α068 13.66 α069 14.13 slimy on plate α070 13.94 α071 13.60 α07212.81 α073 13.10 α074 12.06 α075 9.29 α076 10.86

2 ml culture was inoculated into 30 ml B10 seed medium with 5% glucosein 125 ml baffled flask. It was incubated at 200 rpm for 40 hrs and at300 rpm until 72 hrs.

The following table indicates that the mutants (i.e., α062, α063, andα065) produced more than 20% biomass than that of original X287 slimemutant.

OD₆₀₀ pH DW(g/L) 1 α062 11.5 5.72 14.0 +24% (white slime) 2 α063 12.65.71 13.9 +23% (white slime) 3 α065 11.1 5.57 14.3 +27% (white slime) 4α016 12.5 5.71 13.1 +16% (white slime) 5 X287 10.1 5.73 11.3 Base(Original slime)

The components of the culture medium were as follows:

B10Medium Seed Production Glucose 30 g/L 45 g/L NH₄NO₃ 1 g/L 1.25 g/LMSP* 1 g/L 1.00 g/L K₂HPO₄ 3.2 g/L 0.50 g/L KH₂PO₄ 1.6 g/L MgSO₄* 0.2g/L 0.05 g/L Trace minerals* 1 ml/L 1 ml/L 4% Deformer* 1.5 ml/L DIWater 1 L 1 L

×1000 Trace Minerals ×1000 conc 2703 mg FeCl₃—6H₂O 270.3 g/mol 10 mM1363 mg ZnCl₂ 136.3 g/mol 10 mM 1979 mg MnCl₂—4H₂O 197.9 g/mol 10 mM 238 mg CoCl₂—6H₂O 237.93 g/mol   1 mM  242 mg Na₂MoO₄—2H₂O 241.95g/mol   1 mM  250 mg CuSo₄—5H₂O 249.7 g/mol  1 mM to 1000 ml divide to 4in 250 ml

Example 3 Comparison of Exopolysaccharide Productions Between aSphingomonas Strain No Longer Responsive to SpsN and its Parent Strains

The original slime strain X287 and α016 were compared with mutant α062under several 42 L fermentation tests. 400 ml 24 hrs flask culture wasprepared as 1% starting culture broth for those experiments. The strainATCC31464, parent of X287, was used a capsule forming gellan wild typestrain.

42L/70L-fermentation test (40 g/L Glucose) 24 hrs 48 hrs 60 hrsX287(G116) 11.0 g/L-7980 cp 21.8 g/L-16,000 cp α016(G118)  9.6 g/L-5040cp 19.2 g/L-10,400 cp 20.7 g/L-11,900 cp α062(G120) 23.2 g/L-13,500 cpα062(G131) 10.3 g/L-6650 cp 22.8 g/L-13,100 cp

42L/70L-fermentation test (45*-50 g/L Glucose) 24 hrs 48 hrs 60 hrs 72hrs α062(G121) 6.6 g/L-4160 cp 21.6 g/L-12,300 cp 26.1 g/L-18,000 cpα062(G123) 8.8 g/L-5210 cp 21.7 g/L-15,800 cp 25.5 g/L-24,700 cpα062(G131) 6.9 g/L-3910 cp 23.2 g/L-12,800 cp 25.7 g/L-16,200 cp

The broth was precipitated by 2 volumes IPA and subsequently dried at60° C. for 6 hours. The final viscosity was 4-12 rpm.

The fermentation scores (biomass yield) are as follows.

X287 Duration Glucose Biomass residual Glc broth viscosity G115 70 hrs40 g/L 18.4 g/L   0 g/L 16,400 cp G116 48 hrs 40 g/L 21.8 g/L 0.2 g/L16,000 cp G100 70 hrs 50 g/L 22.9 g/L 4.8 g/L 27,700 cp

α016 Time Glucose Biomass residual Glc broth viscosity G118 60 hrs 40g/L 20.7 g/L 0.6 g/L 11,900 cp

α062 Time Glucose Biomass residual Glc broth viscosity G120 48 hrs 40g/L 23.1 g/L   0 g/L 13,500 cp G131 48 hrs 40 g/L 22.8 g/L   0 g/L13,100 cp G132 48 hrs 40 g/L 22.7 g/L 0.5 g/L 14,400 cp G127 53 hrs 40g/L 23.7 g/L 1.3 g/L 13,700 cp G121 72 hrs 50 g/L 26.1 g/L 2.5 g/L18,000 cp G122 71 hrs 50 g/L 25.9 g/L 4.5 g/L 19,900 cp G128 60 hrs 45g/L 25.7 g/L 1.9 g/L 17,500 cp G139 72 hrs 45 g/L 26.3 g/L 1.9 g/L18,200 cp G141 59 hrs 45 g/L 26.4 g/L   0 g/L 15,300 cp G158 57 hrs 45g/L 26.4 g/L 0.8 g/L 15,800 cp G161 72 hrs 45 g/L 26.2 g/L 0.5 g/L21,700 cp

residual ATCC31461 Glucose Biomass Glc broth viscosity G157 72 hrs 45g/L 22.1 g/L 4.3 g/L 48,100 cp

Culture Broth of α062(G158) was compared with that of ATCC31461(G157).The results are shown in the following table.

(45 g/L glucose) using 70L-fermentor Final Broth α062 (G158) ATCC31461(G157) Frozen G149F 061501C Total Biomass (g/L) 26.4 (59.7%) 22.3(54.8%) Residual Glucose (g/L) 0.8 4.3 Breach debris (g/L) (PHB)  9.1(20.6%)  6.8 (16.7%) Duration (hrs) 57 72 pH (—) 6.7 5.17 OD₆₀₀ (—) 23.427.3 Viscosity (# 4 12-60 rpm) 15,800-3620 48,100-ND Living cells(cells/ml) 1.6 × 10¹⁰ 9.0 × 10⁸ (not accurate) 5% KOH consumption (ml)1270 >1690 Broth appearance Homogeneous slime Not homogeneous Broth DO(%) hit 0% None 36~39 hrs

Example 4 Identification of Xanyhomonas No Longer Responsive to SpsN

The DNA sequence contains biopolymer suppression factor in X029 (ATCCPTA-5127) was transconjugated into Xanthomonas campestris X55(ATCC13951)and X59 (ATCC 55298) by mating. Polymer production suppressed colonieswere isolated as α287, α300 and α301 on tetracycline containingselection plate.

This Xanthan Gum production suppressed α300 (X59:X029) transconjugantwas treated with chemical mutagen EMS. Mutant colonies that do notrespond to suppression factor X029 even under drug pressure wereisolated first then drug sensitive mutants α449 (ATCC PTA-5064), α474,α485, α499, α501, α525, α526, α544 were selected under no drug pressureto get rid of the X029 suppression factor. Those mutants were incubatedin 26 ml YM plus 3& glucose medium using 125 ml baffled flask for 48hrs. Those mutants performance were shown in the following table.

Comparison between X59 and its mutants 0/24/32/48 0/24/32/48 24/32/48hours OD₆₀₀ pH DW (g/L) 1 α449 0.31/7.86/7.62/9.24 6.27/7.28/6.87/5.439.8/12.0/16.4 Ave = 16.0 2 α474 0.27/6.70/6.19/5.79 6.31/7.48/7.04/6.379.9/11.2/15.6 3 α485 0.29/6.82/6.60/7.10 6.26/7.23/7.03/5.947.1/11.3/16.0 4 α499 0.28/6.99/6.43/6.34 6.25/7.40/7.07/6.108.1/10.8/15.7 5 α501 0.26/7.13/6.73/5.13 6.32/7.22/7.04/6.237.7/12.2/16.2 6 α525 0.28/6.99/6.84/8.34 6.29/7.32/7.09/6.017.5/11.0/16.3 7 α526 0.30/7.59/7.22/7.54 6.24/7.33/6.99/5.717.7/11.6/16.5 8 α544 0.24/7.52/5.99/4.99 6.28/7.27/7.11/6.577.3/9.3/15.5 9 X59 0.27/7.28/6.74/6.17 6.24/7.24/7.03/5.93 7.4/10.0/15.3Ave = 15.4 10 X59 0.27/7.21/6.94/7.02 6.23/7.29/7.00/5.67 8.1/9.7/15.5OD₆₀₀(0/24/32/48) Living cell#(0/24/32/48) 1 α449 0.31/7.86/7.62/9.243.9 × 10⁸/8.9 × 10⁹/8.4 × 10⁹/1.0 × 10¹⁰ 2 α474 0.27/6.70/6.19/5.79 3.9× 10⁸/1.3 × 10¹⁰/4.6 × 10⁹/8.0 × 10⁹ 3 α485 0.29/6.82/6.60/7.10 3.7 ×10⁸/9.3 × 10⁹/1.2 × 10¹⁰/8.4 × 10⁹ 4 α499 0.28/6.99/6.43/6.34 4.5 ×10⁸/6.9 × 10⁹/5.7 × 10⁹/7.4 × 10⁹ 5 α501 0.26/7.13/6.73/5.13 4.0 ×10⁸/1.0 × 10¹⁰/1.0 × 10¹⁰/7.4 × 10⁹ 6 α525 0.28/6.99/6.84/8.34 3.7 ×10⁸/9.9 × 10⁹/6.0 × 10⁹/9.5 × 10⁹ 7 α526 0.30/7.59/7.22/7.54 4.6 ×10⁸/1.0 × 10¹⁰/1.4 × 10¹⁰/ND 8 α544 0.24/7.52/5.99/4.99 3.7 × 10⁸/7.8 ×10⁹/6.6 × 10⁹/1.3 × 10¹⁰ 9 X59 0.27/7.28/6.74/6.17 3.6 × 10⁸/8.1 ×10⁹/9.0 × 10⁹/6.9 × 10⁹ 10 X59 0.27/7.21/6.94/7.02 4.6 × 10⁸/8.7 ×10⁹/6.6 × 10⁹/6.8 × 10⁹

α449 and α501 generated 10-20% more biomass than that of parent X59 in32 hrs even with the same level of living cell number. This indicatesthat polymer accumulation speed of those mutants is faster than that ofparent X59.

FIG. 1 shows relation between living cell number and OD₆₀₀ of α449culture. To achieve optimum seed culture, cell numbers were maximizedbased upon OD₆₀₀ value for next stage of production fermentation.

FIG. 2 shows α449 42 L production fermentation using SEB-022-SF+MSPmedium. The open circle represents xanthan gum production fromfermentation of X59 or α449 in SEB-022-MSP in a 6 L fermentor; the solidcircle represents biomass (including cells and xanthan gum) offermentation of α449 using SEB-022-SF+MSP (also referred to as “SFM”) ina 70 L fermentor; and the triangle represents xanthan gum productionfrom fermentation of ATCC13951. The data of the ATCC13951 fermentationin FIG. 2 is from Letisse et al., Applied Microbiology and Biotechnology55(4): 417-22, 2001. MSP refers to digested soy protein.

The final production yield was 57.3 g/L out of 80 g/L sucrose. Thismutant did not respond to suppression factor X029 and accumulated higherlevel of Xanthan gum under the conditions of 28° C., dissolved oxygen(DO) at 30%, and pH6.5.

The α449 400 ml seed culture was inoculated into SEB-022-SF+MSP (Suc=80g/L) medium and incubated for 70 hrs using 70 L fermentor. This mediumcontains 0.69 g/L total nitrogen (0.6 g/L from Soy and 0.09 g/L fromMSP). The final broth OD was 11.3, pH was 6.55, and the living cellnumber was 2.0×10¹⁰/ml. The final broth viscosity was 34,900 cp/16,300cp/9,420 cp-12 rpm/30 rpm/60 rpm using No. 4 spindle by Brookfieldviscometer.

The components of SEC-022-SF+MSP medium used in this example are asfollows:

Sucrose 80.0 g/L MSP 1.0 g/L (N = 0.09) MgSO₄ 0.5 g/L K₂HPO₄ 2.0 g/L SoyProtein 7.6 g/L (N = 0.6) 4% DF289 1 ml/L Trace 1 ml/L

The components of SEC-022-MSP medium used in this example are asfollows:

Sucrose 60.0 g/L MSP 1.0 g/L (N = 0.09) MgSO₄ 0.5 g/L K₂HPO₄ 2.0 g/L SoyProtein 7.6 g/L (N = 0.6) 4% DF289 1 ml/L Trace 1 ml/L

1-45. (canceled)
 46. A method for making bacteria that are capable ofhyper-producing an exopolysaccharide in slime form, comprising: (A)contacting (a) a mutagen, with (b) bacteria that are capable ofproducing an exopolysaccharide in slime form, wherein the bacteria (i)contain a recombinant expression vector comprising at least one promoteroperably linked to a nucleic acid molecule that encodes at least onepolypeptide, wherein said nucleic acid molecule is a nucleotide sequenceaccording to any one of claims 2 to 6, and (ii) express said at leastone polypeptide of (i) such that exopolysaccharide production issuppressed, under conditions and for a time sufficient to producemutagenized bacteria; and (B) identifying among said mutagenizedbacteria one or a plurality of bacteria that are capable ofhyper-producing exopolysaccharide in slime form.
 47. The method of claim46 wherein said mutagen is ethylmethane sulfonate.
 48. The methodaccording to claim 46 wherein said bacteria are Sphingomonas bacteria.49. The method according to claim 48 wherein said Sphingomonas bacteriaare capable of producing an exopolysaccharide selected from the groupconsisting of gellan, welan, rhamsan, diutan, alcalan, S7, S88, 5198,and NW11.
 50. The method according to claim 46 wherein said bacteria areXanthomonas bacteria. 51-53. (canceled)